Identification and characterization of novel long‐term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC‐CI‐MS/MS

Abstract

The search for metabolites with longer detection times remains an important task in, for example, toxicology and doping control. The impact of these long-term metabolites is highlighted by the high number of positive cases after reanalysis of samples that were stored for several years, e.g. samples of previous Olympic Games. A substantial number of previously alleged negative samples have now been declared positive due to the detection of various long-term steroid metabolites the existence of which was unknown during the Olympic Games of 2008 and 2012.In this work, the metabolism of oxymesterone and mesterolone, two anabolic androgenic steroids (AAS), was investigated by application of a selected reaction monitoring gas chromatography-chemical ionization-triple quadrupole mass spectrometry (GC-CI-MS/MS) protocol for metabolite detection and identification. Correlations between AAS structure and GC-CI-MS/MS fragmentation behaviour enabled the search for previously unknown but expected AAS metabolites by selection of theoretical transitions for expected metabolites. Use of different hydrolysis protocols allowed for evaluation of the detection window of both phase I and phase II metabolites.For oxymesterone, a new metabolite, 18-nor-17β-hydroxymethyl-17α-methyl-4-hydroxy-androst-4,13-diene-3-one, was identified. It was detectable up to 46 days by using GC-CI-MS/MS, whereas with a traditional screening (detection of metabolite 17-epioxymesterone with electron ionization GC-MS/MS) oxymesterone administration was only detectable for 3.5 days.A new metabolite was also found for mesterolone. It was identified as 1α-methyl-5α-androstan-3,6,16-triol-17-one and its sulfate form after hydrolysis with Helix pomatia resulted in a prolonged detection time (up to 15 days) for mesterolone abuse.

Gas chromatography−mass spectrometry analysis of non‐hydrolyzed sulfated steroids by degradation product formation

“…Traditionally, mesterolone abuse in routine doping control is detected by GC–MS screening of glucuronidated mesterolone metabolites after hydrolysis with β‐glucuronidase ( E. coli ) . Previous work pointed out that by using this approach G1, mestM20 and mestM22 had the longest detection times . In the excretion studies in this work, GC–MS and LC−TQMS analysis of non‐hydrolyzed sulfated metabolites resulted in longer detection times (factor 2–3.5) in comparison with glucuronidated metabolites on GC–MS (Figure ).…”

“…30,44 Previous work pointed out that by using this approach G1, mestM20 and mestM22 had the longest detection times. 13 In the excretion studies in this work, GC-MS and LC−TQMS analysis of non-hydrolyzed sulfated metabolites resulted in longer detection times (factor 2-3.5) in comparison with glucuronidated metabolites on GC-MS (Figure 3 need to cover all low to medium molecular weight drugs of abuse that need to be detected according to the World Anti-Doping Agency regulations. 47 However, analysis of sulfated steroids on LC-MS requires a specific sample preparation that is not compatible with the wide range of doping substances that need to be detected with the LC-MS screening method.…”

“…In doping control, there has recently been increased interest in the sulfated anabolic androgenic steroid (AAS) fraction. Although only a very small portion of AAS is excreted as sulfates, some of these sulfated metabolites are excreted for a long time in urine and are therefore excellent targets for long‐term detection of AAS abuse . Enabling longer detection times remains an important task and the introduction of new long‐term metabolites for exogenous AAS, such as stanozolol, metandienone, and chlorodehydromethyltestosterone, led to a 4–80‐fold increase of positive findings .…”

“…excreted as sulfates, some of these sulfated metabolites are excreted for a long time in urine and are therefore excellent targets for longterm detection of AAS abuse. 13 Enabling longer detection times remains an important task and the introduction of new long-term metabolites for exogenous AAS, such as stanozolol, metandienone, and chlorodehydromethyltestosterone, led to a 4-80-fold increase of positive findings. [14][15][16][17][18] More than 10 years ago, a long-term metabolite of metandienone was discovered.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…Similarly, the identification of new long‐term metabolites concerning oxymesterone and mesterolone, namely 18‐nor‐17β‐hydroxymethyl‐17α‐methyl‐4‐hydroxyandrost‐4,13‐dien‐3‐one and 1α‐methyl‐5α‐androstane‐3,6,16‐triol‐17‐one, respectively, enabled a significant extension of the drugs' detection windows. Metabolites were predicted in silico, and by means of highly sensitive GC–MS/MS analyses, their existence was eventually proven employing chemical ionization (CI) and MS/MS characterization …”